Rickettsiae are intracellular pathogenic bacteria responsible for various diseases on Humans and animals. Rickettsiae are transmitted by arthropods, most frequently ticks, lice and mites, and cause major illnesses such as epidemic typhus or Rocky Mountain spotted fever. The genus Ehrlichia comprises other species pathogenic for humans and mammals such as E. chaffeensis, responsible for Human monocytic ehrlichiosis, E. canis, the causing agent of canine monocytic ehrlichiosis.
Another species, Ehrlichia ruminantium, formerly known as Cowdria ruminantium, is the causing agent of heartwater or cowdriosis, an economically important disease of domestic ruminants. Heartwater can cause up to 80% mortality in susceptible animals. E. ruminantium is transmitted by Amblyomma ticks and is present in Sub-Saharan Africa and surrounding islands, including Madagascar. Heartwater is also present in several Caribbean islands and is threatening the American mainland.
Vaccination against heartwater has long been based on “infection and treatment”. Naïve animals are inoculated with blood containing virulent organisms, a procedure which carries a high risk of uncontrolled clinical reactions and the inadvertent spread of undesirable parasites and viruses. A first generation cowdriosis inactivated vaccine based on cell-cultured derived elementary bodies was developed. Although representing a considerable improvement and the first heartwater vaccine acceptable for widespread use, the level of protection conferred is still not fully satisfactory. Indeed, all animals develop a clinical reaction at challenge despite vaccination. Furthermore, livestock also faces challenge by genetically and antigenically diverse strains.
Diversity of E. ruminantium is a key problem which has been recognized for a long time, but insufficient information is available for optimum vaccine formulation and specific diagnostic. Serological diagnostic tests of heartwater using crude antigens from whole bacteria detect false positive reactions due to common antigenic determinants
The diversity of E. ruminantium was demonstrated at the antigenic level by cross-immunisation studies. Variable antigens were identified by ELISA and immunoblot using cross-absorbed immune sera.
Genetic diversity was later demonstrated when sequencing the Map 1 gene which showed a high degree of sequence heterogeneity concentrated in three hypervariable regions. Genomic polymorphism was also detected using RAPD and RFLP markers. This DNA polymorphism was shown to correlate with antigenic polymorphism.
ELISA-based and serological diagnostics have been developed using the Map 1 and the GroEL (WO 9914233) antigens. Other peptides for serological diagnostic have been described (US 2002004051, US 20020132789, WO 02/066652). Although they have dramatically improved specificity, they still display cross reaction with E. canis and E. chaffeensis. The map1 gene initially considered as a good marker for geographic diversity, was recently shown not to be geographically constrained. Furthermore, the life span of anti-Map 1 antibodies is rather short.
PCR-based diagnostic methods represent methods of choice for the sensitive and specific detection of Ehrlichia in clinically reactive or asymptomatic carrier ruminants, as well as in vectors. However, in the field, hosts and vectors can be co-infested by several parasites and the diversity of pathogen species is further complicated by the existence of extensive intra-species diversity. Thus, it is important to provide means and diagnostic tools allowing not only to identify E. ruminantium but also to differentiate between different strains.
Sequences allowing differential diagnostic of E. ruminantium strain Gardel and E. ruminantium strain Welgevonden have been previously described by the inventors. They have shown, through complete genome sequencing and comparative genomic analysis that several genes were only found in either strain Gardel or strain Welgevonden, without counterpart in the other strain, and that several other genes, while being present in both strains differed between them by one or several mutations, such as large insertions and/or deletions that result in a frameshift and/or in a truncated version of the original gene. These genes were therefore primary targets to develop specific, multitarget diagnostic methods to differentiate between these two strains (WO 2006/045338; Frutos et al., Journal of Bacteriology. 188: 2533-2542, 2006).